Protein folding is a major unsolved problem in modern biology. A better understanding of the protein folding mechanism will help elucidate why some proteins misfold, leading to disorders like Alzheimers disease, Parkinson's disease, type II diabetes and some cancers (1). In order to understand the role of the environment and cellular conditions in protein folding and what conditions might lead to misfolding, it is important to understand the effect of environmental factors like temperature, solutes and strain. The goal of this proposal is to explore the effect of two environmental factors, force and solutes, on protein folding using optical tweezers. Optical tweezers are used to probe the response of single protein molecules to force along a specific pulling axis (2). Solutes can have large and specific effects on protein processes involving large-scale conformational change (3) and so are a natural perturbant for optical tweezers experiments. Solute effects depend on the amount and type of surface buried or exposed in a process (ASA) and so can be used to probe conformational changes. The experiments outlined here will explore the effects of the denaturant urea and the stabilizing osmolyte glycine betaine (GB) on folding of the src SH3 domain using optical tweezers. 1. Characterize how denaturants and osmolytes affect mechanical folding by developing a folded vs unfolded phase diagram of force vs solute concentration for GB and urea. 2. Explore how folding in single molecule force experiments differs from standard ensemble experiments by determining the effect of urea and GB on folding the src SH3 domain using both techniques. Solute effects are related to ASA for folding and any differences in ASA should reveal differences in the extent of structure in the denatured state ensemble between these experiments. Also, because optical tweezers experiments are performed on single protein molecules, they can be used to look at how behavior of individual proteins deviates from average ensemble behavior, potentially revealing rare folding events that could lead to misfolding. 3. Characterize different folding pathways, by determining the effect of urea and GB on the folding or unfolding rate of the src SH3 domain when pulled along two different axes using optical tweezers. These rates are related to ASA for folding to and from the transition state so can be used to characterize the folding mechanism. Solutes could also potentially perturb the folding process enough to reveal alternate folding pathways.